Properties Of Immobilized Enzymes

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme's catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification.

When an enzyme is immobilized onto a support material, a diffusion layer is created around the particle so that, even with vigorous stirring, substrates must be transported from the bulk of the solution across this stagnant layer to reach the enzyme. Products must also diffuse from the particle surface to the bulk solution. Steric repulsion of substrates and products may also occur, since the enzymesupport system is a crowded molecular environment. Steric factors are particularly significant for enzymes that have high molecular weight substrates, since accessibility of the active site is less easily achieved following immobilization. Particle size is also an important consideration: smaller particles have been observed to yield catalytic properties more closely approximating those obtained with soluble enzymes. The flexibility of polymeric support materials also plays a role in determining the transport properties of substrates and products. Hydrophilic supports have been shown to have less drastic effects on enzyme properties than hydrophobic support materials. Ionic groups on polymeric supports have been shown to interact with enzymes, and also to affect local pH in the microenvironment of the polymer network.

Chemical immobilization methods may alter the local and net charges of enzymes, through covalent modification of charged residues such as lysine (NH4), aspartate, and glutamate (COO-). Conformational changes in secondary and tertiary protein structure may occur as a result of this covalent modification, or as a result of electrostatic, hydrogen-bonding or hydrophobic interactions with the support material. Finally, activity losses may occur as a result of the chemical transformation of catalytically essential amino acid residues.

The specific activity of an enzyme almost always decreases on immobilization. The active sites are less accessible to substrate, and the diffusion of substrates and products across the stagnant layer of solution at the particle surface, and within polymer networks, lowers apparent values of Vmax and raises apparent Km values. The activity of an immobilized enzyme should be expressed as specific activity

Image Turkey Clip Art Color

Relative activity (%)

Figure 4.4. Relative activity versus pH for (b) native chymotrypsin (optimum pH 8.6) and for chymotrypsin bound to (a) polyornithine (optimum pH 7.0), (c) ethylene-maleic anhydride copolymer (optimum pH 9.4). [Reprinted, with permission, from L. Goldstein and E. Katchalski, Fresnius's Z. Anal. Chem. 243, 1968, 375-396. ''Use of Water-Insoluble Enzyme Derivatives in Biochemical Analysis and Separation''.© 1968 by Springer.]

(i.e., units per mg protein) rather than activity per unit weight of the support plus protein. The reason is that the quantity of protein immobilized onto a support varies from a few micrograms to hundreds of milligrams per gram of support material, depending on the immobilization method and the support material chosen. The highest specific activities for immobilized enzymes have been achieved with hydro-philic support materials.

The optimum pH for an enzymatic reaction may shift by as much as 3 pH units upon immobilization.24 This shift is a result of both the charge of the support material and the chemical modification of the enzyme. Figure 4.4 illustrates the dramatic shifts that occur in the pH optimum of chymotrypsin, a proteolytic enzyme, following covalent immobilizations onto a polyornithine (positive) carrier and an ethylene-maleic anhydride copolymer (negative).

This shift in the pH optimum can be explained by an uneven distribution of H+ and OH— between the bulk of the external solution and the polyelectrolyte (carrier) phase. Polyornithine, shown in Figure 4.5, possesses side chains with primary amino groups that are used for the covalent attachment of enzyme. Not all of these groups will react, and those that remain underivatized do so either for steric reasons or due to incomplete coupling reaction steps.

The primary amine groups that remain underivatized exist in the protonated form, and these positively charged —NH^ groups attract OH— from the bulk solution. This increases the local [OH—] relative to that in the bulk, so that

[°H ]surface > [OH Ibulk and PHsurface > PHbulk

Figure 4.5. Enzyme immobilized onto a polyornithine carrier.

The apparent pH optimum therefore occurs at lower measured pH values, and this is observed in Figure 4.4 for chymotrypsin: The apparent pH optimum occurs at pH 7.0 instead of the native value of 8.6 following immobilization onto polyornithine.

When chymotrypsin is immobilized onto the ethylene-maleic anhydride copolymer, one acidic group is created on the surface for each enzyme molecule immobilized, as shown in Figure 4.6. This and other negatively charged supports attract H+ from the bulk of the solution to the surface of the carrier, so that the local pH at the surface is lower

[H+]surface > [H+]bulk and pHsurface < pHbulk

Because the local pH at the surface of the support is lower than the bulk, or measured pH, the apparent pH optimum shifts to higher pH values with this and other negatively charged support materials. Figure 4.4 shows that chymotrypsin immobilized onto an ethylene-maleic anhydride support exhibits a pH optimum of 9.4, almost one full pH unit higher than that observed for the native enzyme.

The increase in apparent Km values observed following the immobilization of enzymes is also readily explained by considering local effects at the carrier surface. Recalling the Michaelis-Menten equation (v = VmaJSVjKm + [S]}), and its derivation (Chapter 2), we know that for soluble enzymes, Km is independent of enzyme concentration and is a constant under a given set of conditions. Immobilized enzymes suspended in an aqueous medium have an unstirred solvent layer surrounding them, called the Nernst or diffusion layer. Substrates and products must diffuse across this layer, and, as a result, a concentration gradient is established for both substrates and products, as shown in Figure 4.7.

Figure 4.6. Enzyme bound to an ethylene-maleic anhydride carrier.

Figure 4.6. Enzyme bound to an ethylene-maleic anhydride carrier.


Figure 4.7. Relative substrate (a) and product (b) concentrations as a function of distance from the surface of a support particle. [S]2bulk > [S]1 bulk and [P]bulk = 0.


Figure 4.7. Relative substrate (a) and product (b) concentrations as a function of distance from the surface of a support particle. [S]2bulk > [S]1 bulk and [P]bulk = 0.

The effects of this concentration gradient are most significant at low bulk concentrations of the substrate, since substrate is converted to product as soon as it reaches the surface of the particle, so that the surface concentration of substrate is zero. At very high bulk substrate concentrations, the enzymatic reaction rate is limited by enzyme kinetics rather than mass transport, so that surface concentrations do not differ significantly from those in the bulk. Because of the concentration gradient, however, enzyme saturation with substrate occurs at much higher bulk substrate concentrations than required to saturate the soluble enzyme. Apparent Km values (Km) for immobilized enzymes are larger than Km values obtained for the native soluble enzymes.

Diffusion layer thickness may be reduced by using smaller particles or by increasing the rate of stirring of the solution; Km values will then approach the

Km values observed for the soluble enzyme. Remember that electrostatic and steric factors may also affect Km values if they lead to local concentrations that differ from bulk concentrations.

The selectivities of enzymes that catalyze reactions involving high molecular weight substrates have been found to change when these enzymes are immobilized, because the diffusion of macromolecular substrates is slower, and because steric factors lower the activity of the enzyme by preventing free access of substrate to the enzyme's active site. For example, the enzyme ribonuclease (RNase) catalyzes the hydrolytic cleavage of phosphodiester bonds linking the nucleotides of polymeric RNA (Eq. 4.20):

RNase will also catalyze phosphodiester bond cleavage in low molecular weight substrates, such as cyclic cytidine monophosphate (cCMP) to produce 5'-CMP (Eq. 4.21):

Agarose Borohydride

Following covalent immobilization onto the hydroxyl groups of agarose, RNase showed decreased activity toward RNA cleavage, when compared to the soluble enzyme.25 In order to make the comparison, it was assumed that the rate of cCMP cleavage would not be affected significantly by the immobilization. Normalized rates could then be calculated as rates of RNA cleavage divided by the rate of cCMP cleavage. These rates were measured for RNA substrates over a wide range of molecular weights (MW), and an empirical correlation was observed between lower reaction rates and higher molecular weights:

normalized rate with soluble enzyme

normalized rate with immobilized enzyme

This study definitively showed the effects of steric exclusion on reaction rates observed with immobilized enzymes.

Enzyme stability with respect to both storage time and denaturation temperature has generally been found to improve upon immobilization. Immobilized enzyme

Relative activity (%)

Incubation time at 75°C (min)

Figure 4.8. Thermal stability of soluble (a) and nylon-immobilized (b) urease at pH 7.0. [Reprinted, with permission, from P. V. Sundaram, and W. E. Hornby, FEBS Letters 10, October 1970, 325-327. ''Preparation and Properties of Urease Chemically Attached to Nylon Tube''. © 1970 by Elsevier.]

reactors may be used for several months with little change in conversion efficiencies. Urease immobilized onto a nylon membrane has been studied with respect to its thermal denaturation properties. Figure 4.8 illustrates the comparative thermal stabilities of immobilized and soluble urease, determined by measuring relative activity as a function of incubation time at elevated temperatures.26 Clearly, the immobilized urease exhibits improved thermal stability that results from the protective microenvironment at the surface of the support.

A spectacular example of stability enhancement through immobilization has been reported for the enzyme catechol-2,3-dioxygenase.27 This enzyme, isolated from the thermophilic bacterium Bacillus stearothermophilus, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde (which can be monitored by absorbance at 375 nm). The soluble enzyme exhibits maximal activity at 50 °C, but following immobilization on glyoxyl agarose beads with a borohydride reduction step, the optimum reaction temperature shifted to 70 °C. At a total protein concentration of 0.010 mg/mL and a temperature of 55 °C, the half-life of the soluble enzyme was 0.08 h, while the enzyme-modified beads had a half-life of 68 h. This represents a 750-fold enhancement of stability that has been attributed to the prevention of subunit dissociation upon immobilization.

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  • Taylor
    Why enzyme immobilized ph higher shift?
    8 years ago
  • albino
    What are the properties of immobilized enzymes?
    3 years ago
  • KY
    What are the properties of immobilized enzyme?
    2 years ago

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